Increased yield of high-purity and active tetrameric recombinant human EC-SOD by solid phase refolding.
Superoxide dismutase (SOD) removes damaging reactive oxygen species from the cellular environment by catalyzing the dismutation of two superoxide radicals to hydrogen peroxide and oxygen. Extracellular superoxide dismutase (EC-SOD) is a tetramer and is present in the extracellular space and to a lesser extent in the extracellular fluids. Increasing therapeutic applications for recombinant human extracellular superoxide dismutase (rEC-SOD) has broadened interest in optimizing methods for its purification, with a native conformation of tetramer. We describe a solid phase refolding procedure that combines immobilized metal affinity chromatography (IMAC) and gel filtration chromatography in the purification of rEC-SOD from Escherichia coli. The purified rEC-SOD tetramer from the Ni(2+)-column chromatography is refolded in Tris buffer. This method yields greater than 90% of the tetramer form. Greater than 99% purity is achieved with further purification over a Superose 12PC 3.2/30 column to obtain the tetramer and specific activities as determined via DCFHDA assay. The improved yield of rEC-SOD in a simple chromatographic purification procedure promises to enhance the development and therapeutic application of this biologically potent molecule.
Ryu
Kang
K
Laboratory of Dermato-Immunology, College of Medicine, The Catholic University of Korea, Seoul 130-701, Korea.
Kim
Young-Hoon
YH
Kim
Younghwa
Y
Lee
Joon-Seok
JS
Jeon
Byeongwook
B
Kim
Tae-Yoon
TY
D000595:Amino Acid Sequence; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005110:Extracellular Space / Q000737:chemistry / Q000201:enzymology; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D011994:Recombinant Proteins; D013482:Superoxide Dismutase